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World Journal of Pharmaceutical Research
Othman et al.
World Journal of Pharmaceutical
Research
SJIF Impact
Factor 5.990
Volume 5, Issue 1, 87-93.
Research Article
ISSN 2277– 7105
GROWTH HORMONE GENE IN IRAQI AND TURKISH AWASSI
SHEEP USING PCR-RFLP
Luma A. Othman1*, Amina N. Althwani1 Abdul Jabbar A.H. Alkhazraji2
1
Genetic Engineering and Biotechnology Institute for Postgraduate Studies, Baghdad
University.
2
Ministry of Science and Technology, Agricultural Researches Directorate.
Article Received on
08 Nov 2015,
Revised on 29 Nov 2015,
Accepted on 19 Dec 2015
ABSTRACT
The objective of the study was to examine the polymorphic GH/HaeIII
gene in Awassi sheep breed (Iraqi and Turkish) Eighty genomic DNAs
that consisted from 40 Iraqi and 40 Turkish Awassi sheep were
isolated from blood samples of those animals. A fragment of
*Correspondence for
GH/HaeIII gene, comprising of a part of intron 2, complete exon 3,
Author
complete intron 3, complete exon 4 and a part of intron 4, was
Luma A. Othman
amplified. The amplified product with the length of 934 bp was
Genetic Engineering and
Biotechnology Institute
digested with HaeIII restriction enzymes and in Turkish Awassi
for Postgraduate Studies,
showed the presence of GH HaeIII homozygotes AA genotype, with
Baghdad University.
fragments 277, 202, 110, 100, 94, 68, 49 and 22,8,4 did not appeare on
agarose gel under Uv light, Homo And heterozygous genotype with
fragments 277,256,202,110,100,94,68,49bp showed in Iraqi Awassi sheep. The present study
concluded that GH/HaeIII could be a genetic marker in sheep for improvement the purity of
the Turkish breed. And diversity in the Iraqi Awassi sheep.
KEYWORDS: Awassi sheep, HaeIII enzyme, homozygosity, hetrozygosity, growth
hormone.
INTRODUCTION
Awassi is the most common breed of sheep in the east of Mediterranean. It is the main sheep
breed in Iraq and Syria, the only native breed in Jordan and Palestine (Hailat, 2005) and
represents an important contribution to sheep breeds in Turkey 3.5% of total sheep population
(Gürsoy, 2005).
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World Journal of Pharmaceutical Research
Awassi sheep is a highly productive indigenous dairy breed as well as producing wool and
meat (Azawi and Al-Mola, 2010).
Growth hormone (GH) is an anabolic hormone synthesized and secreted by the somatotroph
cells of the anterior lobe of the pituitary in a circadian and pulsatile manner, it is a
polypeptide hormone of about 22kDa molecular weight, composed of 190 or 191 amino acids
(Ayuk and Sheppard, 2006).
It plays an important role in body growth and metabolism through protein synthesis, protein
deposition and fat catabolism in tissues and organs and leads to increased nitrogen retention
(Hart and Johnson, 1986; Gluckman etal., 1987), decreased energy retained as fat,
gluconeogenesis and cell division (Eisemann etal., 1986; Neathery etal., 1991).
Growth hormone gene is encoded by 1800 base pairs (bp), consisting of five exons, separated
by four intervening sequences (Gordon etal., 1983).
In the ovine, two alleles of the GH gene have been described. The Gh1 allele contains a
single gene copy (GH1), whereas in the Gh2 allele the gene is duplicated (copies GH2-N and
GH2-Z) with the two copies being located 3.5 kb apart (Valinsky etal., 1990).
Sequence differences between the GH2-N and GH2-Z copies have been demonstrated and
polymorphisms have been found in oGH coding and non-coding regions (Ofir and Gootwine,
1997).
This study shed light on the polymorphic of the GH/HaeIII gene in Awassi sheep breed (Iraqi
and Turkish).
MATERIALS AND METHODS
Blood collection
Eighty blood samples collected from (40) Iraqi and (40) Turkish Awassi sheep of different
ages (male and female) took from ministry of science and technology/agricultural researches
(Al-Zaafarania,20 km south of Baghdad) and from ministry of agriculture/research station of
sheep and goat (Abu-Ghraib,25km west of Baghdad), from November 2014 to April 2015 at
university of Baghdad/Institute of genetic engineering and biotechnology for post graduate
studies.
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World Journal of Pharmaceutical Research
Blood samples were collected from jugular vein by vacutainer needle into 6 ml tubes
containing 1 ml of 10% ethylene diamine tetra acetic acid (EDTA). The samples were stored
in cool box containing ice jelly pack during collection process then kept at (-20oC) until ready
for DNA isolation.
DNA isolation and amplification
The genomic DNAs were successfully isolated from the blood samples using DNA extraction
kit (Bioneer, genomic DNA isolation kit, korea).
Polymerase Chain Reaction (PCR) was carried out using Eppendorf thermocycler (Biometra,
Germany) and PCR Green Master Mix (Promega Corporation, USA) containing bacterially
derived Taq DNA polymerase, dNTPs, MgCl2 and reaction buffers at optimal concentrations
for efficient amplification of DNA templates by PCR. Each reaction mixture consisted of
12.5 µl of taq green PCR master mix, 5 μl of the DNA solution (50 to 100 ng/μl), 1 μl of each
primer (10 pmol/μl) and 5.5 µl free nuclease water .Amplification for a 934 bp fragment from
the intron II to the intron IV of the oGH gene was carried out using primer according to
Kuulasma (2002) as follows.
GH-F: 5’-GGAGGCAGGAAGGGATGAA-3’ and
GH-R: 5’-CCAAGGGAGGGAGAGACAGA-3’.
The amplification was carried out using thermolcycler (Eppendorf Mastercycler) with the
following conditions: initial denaturation at 95°C for 5 min, by 33 cycles of denaturation at
95°C for 45s; annealing at 60°C for 45s and extension at 72°C for 45s followed by a final
extension at 72°C for 10 min. Each amplified product was analyzed by electrophoresis on a
2% (w/v) agarose gel, using ethidium bromide.
RFLP analysis and electrophoresis
Restriction fragment length polymorphism (RFLP) analysis was conducted to detect
polymorphism sites. The PCR amplicons were digested with HaeIII restriction endonuclease
(Bio Labs Inc, New England) The amount of digestion mixture was 5.7 µl free nuclease
water, 3 µl Digest buffer, 1 µl HaeIII restriction enzyme, 0.3 µl from bovine serum albumine
(BSA) with 20 µl PCR DNA product. The reaction mixture was digested in a thermocycler
at 37oC for 4 h., then twenty µl of samples and then detected by 3.5% agarose gel
electrophoresis. The electrophoresis product restriction by HaeIII enzyme was interpreted by
comparing them with 50bp marker band.
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RESULTS AND DISCUSSION
Amplification of growth hormone gene
This study throw light on the awassi sheep that consider the most important breed in Iraq and
high light on the importance of the gene that coding for growth hormone which in turn
responsible for the meat and milk production in the sheep.
The DNA that extracted from blood sample of Iraqi sheep and also the Fourty Turkish awassi
sheep were used for comparising showed clear sharp bands that presented on agarose gel in
electrophoresis and then examined under Uv light, the purity of these DNAs were perfect
ranged from 1.8-1.9 and the concentration were ranged from 16-20ng/µl according to
Sambrook and Russel (2001).
The PCR product or amplicon of awassi sheep (Iraqi and Turkish) was also sharp and match
to the marker 934 bp and that come in agreement with the oGH gene of lohi breed that
located from intron II to the intron IV (accession no Genbank GQ452268, Gul, etal., 2009)
And Indonesia fat tailed sheep (Donggala and East java breed, Malewa, etal., 2014).
1
2
3
4
5
6
7
8
9
10
11
12
934 bp
Figure (1): PCR products of growth hormone gene with size of 934 bp. The product was
electrophoresis on 2% agarose gel at 5 volt/cm2 for 1hour. Lane 1 DNA ladder (1001000), Lane (2-12) PCR products of the growth hormone gene from Awassi sheep with
size 934bp.visulized under U.V light after stain with Ethidium Bromide.
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The result of restriction enzyme were revealed AA allele (homozygous) that had seven
fragments namely 277,202,110,100,94,68,49bp in Turkish Awassi sheep found and in Iraqi
Awassi AB allele (heterozygous) namely 277,256,202,110,100,94,68,49bp and homozygous
also found this showed the range of purity of the Turkish herd that had been sampling of it,
and it is pure breed and there is inbreeding between them, the probability of homozygous
presence may be due to the adaptation of these breed to live in this region. the diversity in
Iraqi Awassi sheep is marking there is cross breeding between them.
277 bp
202 bp
110 bp
100 bp
94 bp
68 bp
49 bp
Figure (2): PCR product digested with HaeIII restriction enzyme electrophoresis on
3.5%. Lane1: DNA ladder (50 bp). Lane 2, 10: AA allele (homozyguse) of growth
hormone gene. The (RFLP) products at agarose gel at 5 volt/cm2 for 1hour. visulized
under U.V light after stain with Ethidium Bromide.
The result of this study showed that GH/HaeIII had the different in both Iraqi and Turkish
awassi sheep in homo and hetrozygosity and this came in agreement with study of, Malewa
(2014) revealed there was both homo and heterozygots in Indonesia fat tailed sheep
(Donggala and East Java breed) Which had allele AB (277, 256, 202, 110, 100, 94, 68,
49,22,21,8,4) that notes transition in the exon 3 (AG-CC) to (GG-CC) of the gene at 227 base
IV (accession no Genbank GQ452268, Gul, etal .,2009) or at 255 base of PCR product.
Cobra, etal. (2013) suggested that there were association between different growth hormone
genotypes and growth triats including birth weight (BW), weaning weight (WW), six month
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weight (SW), nine month weight (NW) and w12weight (yearling weight) of makooei sheep
by using PCR-SSCP. Gupta etal. (2007) used the PCR-SSCP analysis to investigate growth
hormone polymorphism in Indian black banegal goats, while Bastos etal. (2001) detected two
and five SSCP patterns for exon4 and 5 respectively.
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